Stimulation of sulfate and thymidine incorporation into hypophysectomized rat cartilage by growth hormone and insulin-like growth factor-I in vitro: the somatomedin hypothesis revisited.

These studies were undertaken in an attempt to demonstrate sensitivity of cartilage from hypophysectomized rats to growth hormone (GH) in physiologic concentrations in vitro and to compare the actions of the hormone and extrinsic insulin-like growth factor-I (IGF-I). In a HEPES-buffered, amino acid-glucose nutrient medium with a low concentration of bovine serum albumin, costal cartilage explants from such animals were responsive to purified or recombinant bovine GH (bGH) and recombinant human GH (hGH) with a sensitivity of less than 100 ng/ml [corrected]. Sulfate incorporation into proteoglycans and thymidine incorporation into DNA were increased. The effect on sulfate incorporation was near maximal by 24 hours, but the peak effect on thymidine incorporation was delayed. Comparison of the dose-response relationships of bGH and IGF-I demonstrated a greater effect of IGF-I with increasing concentrations of each. The action of bGH was inhibited by an immunoglobulin G fraction of an IGF-I antiserum. These results are consistent with previously available evidence that IGF-I has an autocrine/paracrine action on cartilage, which is regulated by GH. However, the limited cartilage response to the direct action of GH supports the hypothesis of Daughaday that IGF-I also has an endocrine role in the stimulation of skeletal growth by the pituitary hormone.

Intraocular tumor from disseminated histoplasmosis.

We identified and treated a solid, growing fungal tumor mass in a patient with disseminated histoplasmosis. Although the most commonly reported intraocular lesions from disseminated histoplasmosis are areas of inactive chorioretinal scars or areas of localized subretinal neovascular membrane formation, a focus of active fungal growth needs to be ruled out in all such cases. When a solid tumor mass is identified, the most effective way of preserving vision is with systemic antifungal therapy.

Hypercalcemia in active pulmonary tuberculosis.

We describe four cases of active pulmonary tuberculosis with hypercalcemia. The hypercalcemia developed three to eight weeks after the patients were admitted to the hospital, when clinical improvement due to specific drug therapy for the infection was also evident. The abnormality persisted for three to seven weeks and subsided spontaneously. In each case, the serum level of 1,25-dihydroxyvitamin D determined during the period of hypercalcemia (at the peak in three of the four cases) was low. This suggests that altered vitamin D metabolism is not the basis for hypercalcemia in all cases of pulmonary tuberculosis.

Hypercortisolism, diarrhea with steatorrhea, and massive proteinuria due to pheochromocytoma.

Amelioration or cure of hypertension, hypercortisolism, diarrhea with steatorrhea, and massive proteinuria resulted from excision of a pheochromocytoma that contained immunoreactive ACTH, VIP, and somatostatin. Ectopic ACTH production by the tumor was clearly the cause of the hypercortisolism, and the possible involvement of VIP and somatostatin in the diarrhea and steatorrhea was considered. The response to tumor removal suggested that the mesangioproliferative glomerulonephritis shown on renal biopsy was also a paraneoplastic phenomenon.

Stimulation of uridine, leucine, and sulfate incorporation into rat cartilage macromolecules by adenosine 3',5'-monophosphate.

Conflicting findings on the ability of cAMP analogs or phophodiesterase inhibitors to stimulate precursor incorporation into macromolecules of rat cartilage have been reported. Therefore, the effects of these compounds on the incorporation of uridine into RNA, leucine into proteins, and sulfate into proteoglycans have been reexamined in cartilage from normal and hypophysectomized rats. When cartilage was incubated for 24 h in a medium with the test agents and then pulsed for 2 h in the basal medium containing labeled precursors, both monobutyryl cAMP (BucAMP) and methylisobutylxanthine (MIX) enhanced the ability of the tissue to incorporate precursors into macromolecules. The effect of BucAMP was significant in most cases at a concentration of 30 microM, optimal at concentrations of 100-300 microM, and diminished at a concentration of 1000 microM. Similar stimulation was produced by dibutyrul cAMP [(Bu)2cAMP] or 8-dimethylamino cAMP, but monobutyryl cGMP was ineffective. MIX in a concentration of 20 microM increased precursor incorporation in most cases, and a concentration of 100 microM was optimal; at a concentration of 500 microM, MIX had no significant effect on leucine or sulfate incorporation. When cartilage from hypophysectomized rats was incubated in a medium with the test agents for 4-6 h and the labeled precursors were added for the last 2 h, BucAMP did not increase incorporation of any of the precursors. MIX was also ineffective, even though tissue cAMP levels were increased. However, precursor incorporation was increased by exposure to partially purified rat somatomedin for the same periods. The degree of stimulation of sulfate incorporation induced by either BucAMP or MIX was proportional to the time of exposure to these agents. Preincubation of cartilage in basal medium alone for 22 h or longer increased basal sulfate incorporation, but caused only a slight enhancement of the action of BucAMP. The addition of synthetic bovine PTH-(1-34) (1 microM) to the incubation medium increased sulfate incorporation into hypophysectomized rat cartilage by 24 h, and this effect was potentiated by MIX (10 microM). No stimulation was detectable by 6 h, even with MIX in the medium. PTH-(1-34) increased the cartilage cAMP level, and this effect was also potentiated by MIX. In the presence of MIX, PTH-(1-34) increased the level of cAMP within 30 min, while the rat somatomedin preparation had no effect on the cAMP level during 60 min of incubation. The level of cartilage cGMP was not raised by either PTH or somatomedin.(ABSTRACT TRUNCATED AT 400 WORDS)

Similarity of somatomedin inhibitor in sera from starved, hypophysectomized, and diabetic rats: distinction from a heat-stable inhibitor of rat cartilage metabolism.

The inhibitory effects of sera from starved, hypophysectomized, and alloxan-diabetic rats on basal and somatomedin-stimulated sulfate incorporation into cartilage from hypophysectomized rats were compared. The somatomedin inhibitory activity in serum from diabetic rats behaved like that in serum from starved rats on heating at 60 C. Both were labile in the native sera (pH 8.3-8.4), but activity was conserved to a large extent by lowering the pH to 7.4 and diluting the sera before heating. In all of these sera the peak of the somatomedin inhibitory activity was eluted from a column of Sephacryl S-200 at pH 7.4 just after albumin, and lesser amounts were eluted with albumin and higher molecular weight components. Activity of this type was undetectable in fractions prepared from sera that had been heated at 60 C. These results indicate the similarity of this inhibitor in starved, hypophysectomized, and diabetic rat sera. Certain fractions of both starved and diabetic rat sera, which were eluted from a column of Sephacryl S-200 beyond the total bed volume, contained heat-stable inhibitory activity. In contrast to the effects of the heat-labile inhibitor, these fractions only inhibited basal sulfate incorporation into hypophysectomized rat cartilage under the assay conditions employed. This heat-stable inhibitor was not detected in fractions of hypophysectomized rat serum, and inhibitory concentrations of corticosterone were present in fractions of starved and diabetic rat sera containing the material. The findings suggest that the heat-stable inhibitor is corticosterone.

Effects of cyclic nucleotides on deoxyribonucleic acid synthesis in hypophysectomized rat cartilage: stimulation of thymidine incorporation and potentiation of the action of somatomedin by analogs of adenosine 3',5'-monophosphate or a cyclic nucleotide phosphodiesterase inhibitor.

The actions of cyclic nucleotides on basal and somatomedin-stimulated thymidine incorporation into DNA by costal cartilage from hypophysectomized rats were investigated. Three analogs of cAMP (dibutyryl, 8-bromo, and 8-dimethylamino derivatives, which are alternate activators of cAMP-dependent protein kinase and resistant to degradation by cAMP phosphodiesterase but represent a wide difference in potency as phosphodiesterase inhibitors) in range of concentrations from about 10(-5) to 3 X 10(-4) M enhanced basal and somatomedin-stimulated thymidine incorporation. Each cAMP analog at optimal concentration produced combined effects with a suboptimal concentration of somatomedin which were additive or greater. cAMP itself, 5'-AMP, adenosine, 8-Br-5'-AMP, 8-Br-AMPT, and cGMP at concentrations from 10(-7)--10(-3) M or dibutyryl cGMP at concentrations from 10(-10)--10(-3) M did not reproduce the effects of the cAMP analogs. A phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine) at concentrations of 100 or 500 microM also potentiated the effects of somatomedin. At 100- or 500-microM concentrations, the phosphodiesterase inhibitor increased cartilage levels of cAMP and cGMP. These results suggest a role for cAMP in DNA synthesis in rat cartilage. However, they fail to support the hypothesis that all effects of somatomedin on that process are mediated by cAMP, since stimulation of thymidine incorporation by the hormone can be demonstrated in cartilage maximally stimulated by analogs of cAMP.

Somatomedin and analogues of cyclic AMP increase the number of cells synthesizing DNA in cartilage from hypophysectomized rats.

The effects of somatomedin and certain nucleotides on nuclear labelling of cartilage cells with [3H]thymidine were determined by autoradiography. Segments of costal cartilage from hypophysectomized rats were incubated for 24 h in a basal medium with or without additions and then pulsed for 2 h with [3H]thymidine in the basal medium. Both somatomedin (0.1 U/ml) and Bt2cAMP (10(-4)M) increased the number of labelled nuclei, and the combined effects were more than additive. A parallelism between the effects of these agents on nuclear labelling and their effects on total thymidine incorporation into DNA was demonstrated. The 8-bromated derivative of cAMP (10(-4)M) also enhanced chondrocyte nuclear labelling, but neither 8-Br-5'-AMP (10(-4)7) nor 8-Br-cGMP (10(-4)M) exhibited actions of the cAMP analogues. It is concluded that in cartilage obtained from hypophysectomized rats and incubated under the specified conditions (1) both somatomedin and cAMP analogues increase the number of cells synthesizing DNA as well as total thymidine incorporation into DNA, (2) the effects of the hormone and cyclic nucleotide in combination are synergistic, and (3) the increased incorporation of labelled thymidine into DNA reflects increased DNA synthesis and not merely an alteration of the specific activity of the intracellular thymidine nucleotide pool.

Evidence that endotoxin is the cyclic 3':5'-GMP--promoting factor in erythropoietin preparations.

Since Ep preparations are contaminated with endotoxin, the possibility that the latter might be the factor in crude Ep which increases cGMP levels in rat fetal liver cells was examined. Endotoxin produced a striking elevation of cGMP in rat fetal liver cells without affecting cAMP levels or heme synthesis. Absorption with Limulus lysate of more than 99% of the endotoxin in a crude Ep preparation caused a parallel decrease in the cGMP-promoting activity without reduction of heme synthetic potency. It is concluded that endotoxin is the component of crude Ep which increases cGMP levels in rat fetal liver. The precise role of elevated cGMP in the action of endotoxin on cells and the universality of this effect remain to be determined.

Somatomedin.

Somatomedin is a peptide component of serum which has been postulated to mediate the action of growth hormone on skeletal tissue. Direct effects on cartilage include stimulation of the synthesis of mucopolysacharide, protein, and nucleic acids. Insulin-like effects on non-skeletal tissues and cells have been described, and a relationship to NSILA-S and MSA has been suggested. The liver may be an important source. Non-specificity of bioassays is a problem. Growth hormone deficiency, malnutrition, therapy with corticosteroids or estrogens, and a type of dwarfism characterized by high serum growth hormone are associated with decreased somatomedin. An unexplained phenomenon is the normal somatomedin with low or undetectable growth hormone in certain cases of craniopharyngioma or other tumors involving the hypothalamus. Somatomedin is increased in acromegaly.

Glucocorticoid inhibition of sulfate incorporation by cartilage of normal rats.

The effects of dexamethasone, cortisol, and deoxycorticosterone on sulfate incorporation by cartilage of normal rats were determined. In vivo dexamethasone was a more potent inhibitor than cortisol, and deoxycorticosterone in the dose tested was ineffective. In vitro dexamethasone and cortisol were inhibitory in concentrations of 10-8 and 10-7M, respectively, when sulfate incorporation was measured after 24 h of incubation. Dexamethasone was at least 10 times as potent as cortisol. Deoxycorticosterone was inhibitory in a concentration of 10-4M. It is concluded that inhibition of mucopolysaccharide synthesis in cartilage of normal rats by adrenal cortical steroids or their analogs, both in vivo and in vitro, is correlated with glucocorticoid activity.

Evidence for structural dissociation of two biologic actions of growth hormone.

The effects of purified growth hormone and its CNBr fragments on somatomedin induction and on the stimulation of hepatic and renal ornithine decarboxylase (L-ornithine carboxylase, EC 4.1.1.17) activity in rats have been investigated. At the doses tested, none of the CNBr fragments induced somatomedin as evidenced by lack of an effect on sulfate, leucine, and thymidine incorporation into cartilage of hypophysectomized rats. However, the largest fragment, consisting of two peptides corresponding to Residues 6-124 and 150-179 linked by a disulfide bridge, stimulated both renal and hepatic ornithine decarboxylase activity in hypophysectomized rats and the activity of the hepatic enzyme in intact animals. A smaller CNBr fragment corresponding to Residues 125-149 slightly stimulated the activity of renal ornithine decarboxylase but failed to increase activity of the hepatic enzyme. A similar slight stimulation of the activity of the renal, but not the hepatic, enzyme was produced by a large carboxyl-terminal fragment (molecular weight 8000) prepared by proteolytic cleavage of partially purified ovine growth hormone. Circular dichroic spectra of the CNBr fragments demonstrated that the largest fragment retained much of the ordered secondary structure of intact growth hormone while two smaller CNBr fragments were devoid of ordered secondary structure. These observations indicate that different biological activities of growth hormone may be dissociated by fragmentation of the parent molecule.